U.S. patent application number 11/742664 was filed with the patent office on 2007-08-30 for composition and method for treating fibrotic diseases.
Invention is credited to Gao Yun Hu, Yuenian Eric Shi, Li Jian Tao.
Application Number | 20070203203 11/742664 |
Document ID | / |
Family ID | 37076581 |
Filed Date | 2007-08-30 |
United States Patent
Application |
20070203203 |
Kind Code |
A1 |
Tao; Li Jian ; et
al. |
August 30, 2007 |
Composition and Method for Treating Fibrotic Diseases
Abstract
The present invention discloses 5-methyl-1-(substituted
phenyl)-2(1H)-pyridones have enhanced anti-fibrotic activities than
5-methyl-1-(non-substituted phenyl)-2(1H)-pyridones. An
representative example of 5-methyl-(1-substituted
phenyl)-2(1H)-pyridones is
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone. Accordingly, there are
provided compositions comprising one or more compounds selected
from the group consisting of 5-methyl-1-(substituted
phenyl)-2(1H)-pyridones and methods of using the same to treat or
prevent fibrosis diseases.
Inventors: |
Tao; Li Jian; (Changsha,
CN) ; Hu; Gao Yun; (Changsha, CN) ; Shi;
Yuenian Eric; (Roslyn Heights, NY) |
Correspondence
Address: |
LAW OFFICES OF ALBERT WAI-KIT CHAN, LLC
WORLD PLAZA, SUITE 604
141-07 20TH AVENUE
WHITESTONE
NY
11357
US
|
Family ID: |
37076581 |
Appl. No.: |
11/742664 |
Filed: |
May 1, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60849039 |
Oct 3, 2006 |
|
|
|
Current U.S.
Class: |
514/345 |
Current CPC
Class: |
A61P 13/08 20180101;
A61P 27/06 20180101; A61K 31/4418 20130101; A61K 31/4412 20130101;
A61P 19/08 20180101; A61P 43/00 20180101; Y02A 50/30 20180101; A61P
1/16 20180101; A61P 11/00 20180101; A61P 13/12 20180101; A61P 25/28
20180101; A61P 9/00 20180101; A61P 9/04 20180101; A61P 21/00
20180101; Y02A 50/423 20180101; A61P 9/10 20180101; A61P 17/00
20180101 |
Class at
Publication: |
514/345 |
International
Class: |
A61K 31/4412 20060101
A61K031/4412 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 11, 2006 |
CN |
PCT/CN06/00651 |
Apr 13, 2005 |
CN |
200510031445.7 |
Claims
1. A composition comprising one or more 5-methyl-1-(substituted
phenyl)-2(1H)-pyridone in an amount effective for treating organ or
tissue fibrosis, said 5-methyl-1-(substituted
phenyl)-2(1H)-pyridone having a formula of: ##STR5## wherein n=1 or
2; R is selected from the group consisting of F, Cl, Br, I, nitro,
C.sub.1-C.sub.6 straight-chain alkyl group, C.sub.3-C.sub.6
branched-chain alkyl group, C.sub.1-C.sub.6 straight-chain alkoxy
group, C.sub.3-C.sub.6 branched-chain alkoxy group, and halogenated
C.sub.1-C.sub.6 alkyl group; and when n=2, not both R are
nitro.
2. The composition of claim 1, wherein the 5-methyl-1-(substituted
phenyl)-2(1H)-pyridone is selected from the group consisting of
1-(2'-bromophenyl)-5-methyl-2(1H)-pyridone,
1-(3'-bromophenyl)-5-methyl-2(1H)-pyridone,
1-(4'-bromophenyl)-5-methyl-2(1H)-pyridone, 1-(2'-
chlorophenyl)-5-methyl-2(1H)-pyridone,
1-(3'-chlorophenyl)-5-methyl-2(1H)-pyridone,
1-(2'-fluorophenyl)-5-methyl-2(1H)-pyridone,
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone,
1-(4'-fluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2'-iodophenyl)-5-methyl-2(1H)-pyridone,
1-(3'-iodophenyl)-5-methyl-2(1H)-pyridone,
1-(4'-iodophenyl)-5-methyl-2(1H)-pyridone,
1-(2',3'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(3',5'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(2',3'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(3',5'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',3'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(3',5'-difluorophenyl)-5-methyl-2(1H)-pyridone,
5-methyl-1-(2'-trifluoromethylphenyl)-2(1H)-pyridone,
5-methyl-1-(4'-trifluoromethylphenyl)-2(1H)-pyridone,
1-(2',3'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone,
1-(3',5'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone,
5-methyl-1-(2'-methylphenyl)-2(1H)-pyridone,
1-(3'-methylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',3'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(3',5'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2'-methoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(3'-methoxyphenyl)-5-methyl-2(1H)-pyridone.
1-(2',3'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone, and
1-(3',5'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone.
3. The composition of claim 1, wherein the amount effective for
treating organ or tissue fibrosis comprises a daily dosage of about
25 mg to about 6,000 mg.
4. The composition of claim 1, wherein the amount effective for
treating organ or tissue fibrosis comprises a daily dosage of about
50 mg to about 2000 mg.
5. The composition of claim 1, wherein the amount effective for
treating organ or tissue fibrosis comprises a daily dosage of about
100 mg to about 1000 mg.
6. A pharmaceutical composition comprising the composition of claim
1 and a pharmaceutically acceptable carrier.
7. A method of treating organ or tissue fibrosis, comprising
administering to a subject an effective amount of a composition
comprising one or more 5-methyl-1-(substituted
phenyl)-2(1H)-pyridones having a formula of: ##STR6## wherein n=1
or 2; R is selected from the group consisting of F, Cl, Br, I,
nitro, C.sub.1-C.sub.6 straight-chain alkyl group, C.sub.3-C.sub.6
branched-chain alkyl group, C.sub.1-C.sub.6 straight-chain alkoxy
group, C.sub.3-C.sub.6 branched-chain alkoxy group, and halogenated
C.sub.1-C.sub.6 alkyl group; and when n=2, not both R are
nitro.
8. The method of claim 7, wherein the 5-methyl-1-(substituted
phenyl)-2(1H)-pyridone is selected from the group consisting of
1-(2'-bromophenyl)-5-methyl-2(1H)-pyridone,
1-(3'-bromophenyl)-5-methyl-2(1H)-pyridone,
1-(4'-bromophenyl)-5-methyl-2(1H)-pyridone,
1-(2'-chlorophenyl)-5-methyl-2(1H)-pyridone
1-(3'-chlorophenyl)-5-methyl-2(1H)-pyridone,
1-(2'-fluorophenyl)-5-methyl-2(1H)-pyridone,
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone,
1-(4'-fluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2'-iodophenyl)-5-methyl-2(1H)-pyridone,
1-(3'-iodophenyl)-5-methyl-2(1H)-pyridone,
1-(4'-iodophenyl)-5-methyl-2(1H)-pyridone,
1-(2',3'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(3',5'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(2',3'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(3',5'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',3'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(3',5'-difluorophenyl)-5-methyl-2(1H)-pyridone,
5-methyl-1-(2'-trifluoromethylphenyl)-2(1H)-pyridone,
5-methyl-1-(4'-trifluoromethylphenyl)-2(1H)-pyridone,
1-(2',3'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone, or
1-(3',5'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone.
5-methyl-1-(2'-methylphenyl)-2(1H)-pyridone,
1-(3'-methylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',3'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(3',5'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2'-methoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(3'-methoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(2',3'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone, and
1-(3',5'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone.
9. The method of claim 7, wherein the amount effective for treating
organ or tissue fibrosis comprises a daily dosage of about 25 mg to
about 6,000 mg.
10. The method of claim 7, wherein the amount effective for
treating organ or tissue fibrosis comprises a daily dosage of about
50 mg to about 2000 mg.
11. The method of claim 7, wherein the amount effective for
treating organ or tissue fibrosis comprises a daily dosage of about
100 mg to about 1000 mg.
12. The method of claim 7, wherein the organ or tissue fibrosis is
selected from the group consisting of glomerulus sclerosis, renal
interstitial fibrosis, liver fibrosis, pulmonary fibrosis,
peridoneal fibrosis, myocardiac fibrosis, fibrosis of skin,
post-surgical adhesion, benign prostate hypertrophy,
musculoskeletal fibrosis, scleroderma, Alzheimer's disease,
fibrotic vascular disease, and glaucoma.
13. The method of claim 7, wherein the composition is administered
through a route selected from the group consisting of oral
administration, parenteral administration, nasal administration,
rectal administration, vaginal administration, ophthalmic
application, and topical application.
14. The method of claim 7, wherein the 5-methyl-1-(substituted
phenyl)-2(1H)-pyridone is
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone.
15. The method of claim 14, wherein the subject experiences less
toxicity than treatment with 5-methyl-1-phenyl-2(1H)-pyridone.
16. A compound of 5-methyl-1-(substituted phenyl)-2(1H)-pyridone
selected from the group consisting of
1-(2'-chlorophenyl)-5-methyl-2(1H)-pyridone,
1-(3'-chlorophenyl)-5-methyl-2(1H)-pyridone,
1-(2'-fluorophenyl)-5-methyl-2(1H)-pyridone,
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone,
1-(4'-fluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2'-iodophenyl)-5-methyl-2(1H)-pyridone,
1-(3'-iodophenyl)-5-methyl-2(1H)-pyridone,
1-(4'-iodophenyl)-5-methyl-2(1H)-pyridone,
1-(2',3-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(3',5'-difluorophenyl)-5-methyl-2(1H)-pyridone and
1-(3',4'-dichlorophenyl)-5-methyl-2(1H)-pyridone.
17. A pharmaceutical composition comprising one or more of the
compound of claim 16 and a pharmaceutically acceptable carrier.
18. A method of treating organ or tissue fibrosis, comprising
administering to a subject an effective amount of the composition
of claim 17.
19. The method of claim 18, wherein the amount effective for
treating organ or tissue fibrosis comprises a daily dosage of about
25 mg to about 6,000 mg.
20. The method of claim 18, wherein the amount effective for
treating organ or tissue fibrosis comprises a daily dosage of about
50 mg to about 2000 mg.
21. The method of claim 18, wherein the amount effective for
treating organ or tissue fibrosis comprises a daily dosage of about
100 mg to about 1000 mg.
22. The method of claim 18, wherein the organ or tissue fibrosis is
selected from the group consisting of glomerulus sclerosis, renal
interstitial fibrosis, liver fibrosis, pulmonary fibrosis,
peridoneal fibrosis, myocardiac fibrosis, fibrosis of skin,
post-surgical adhesion, benign prostate hypertrophy,
musculoskeletal fibrosis, scleroderma, Alzheimer's disease,
fibrotic vascular disease, and glaucoma.
23. The method of claim 18, wherein the composition is administered
through a route selected from the group consisting of oral
administration, parenteral administration, nasal administration,
rectal administration, vaginal administration, ophthalmic
application, and topical application.
24. The method of claim 18, wherein the subject experiences less
toxicity than treatment with 5-methyl-1-phenyl-2(1H)-pyridone.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S. Ser.
No. 60/849,039, filed Oct. 3, 2006, International Application No.
PCT/CN2006/000651, filed Apr. 11, 2006, and Chinese application No.
200510031445.7, filed Apr. 13, 2005. The entire contents and
disclosures of the preceding applications are incorporated by
reference into this application.
[0002] Throughout this application, various references or
publications are cited. Disclosures of these references or
publications in their entireties are hereby incorporated by
reference into this application in order to more fully describe the
state of the art to which this invention pertains.
FIELD OF THE INVENTION
[0003] This invention is related to compositions comprising
5-methyl-1-(substituted phenyl)-2(1H)-pyridones and methods of
using the same to treat fibrotic diseases.
BACKGROUND OF THE INVENTION
[0004] Fibrosis can occur in various organs or tissues, causing
reduction of healthy cells within any organ or tissue and increase
in the mass of fibrotic connective tissues, eventually damage the
normal structure of the organs or tissues. The damages can impair
the physiological and biochemical functions of the affected organs
or tissues, and may cause organ shut down completely. The
pathogenesis, diagnostic methods, methods of prevention and
treatment for organ and tissue fibrosis have been studied
extensively. Much progress has been made in certain areas. However
there are still many challenges, especially in the area of
developing effective therapeutics.
[0005] It is generally believed that fibrosis of organs or tissues
are caused by multiple factors such as inflammation, immunological
reactions, ischemia, hemodynamics change etc. that cause
inflammatory denaturing and narcosis of parenchymal cells. The
impaired parenchymal cells in turn activate macrophages to release
numerous cytokines and growth factors, among which TGF-.beta. is a
critical one. TGF-.beta. can activate quiescent extracellular
matrix (ECM) producing cells and turn them into myofibroblast. The
newly formed fibroblasts not only increase production of collagen,
a key protein of ECM, but also decrease destruction of ECM. The net
result is accumulation of extracellular matrix that leads to organ
or tissue fibrosis. Thus, initiation and development of organ or
tissue fibrosis is the results of inflammatory response and
production of inflammatory cytokines, mainly TGF-.beta.. Logically,
due to the crucial role of TGF-.beta. on ECM accumulation and
formation of organ or tissue fibrosis, one of the important goals
in early development or screening of antifibrotic drugs is to find
a way to inhibit the production of pro-inflammatory cytokines,
e.g., TGF-.beta.. However, more convincing data for any
antifibrotic drug candidate will obviously come from the test using
various in vivo fibrotic models.
[0006] A number of compounds that exhibit anti-inflammatory and
anti-fibrotic activities have been described. U.S. Pat. Nos.
3,839,346, 3,974,281, 4,042,699 and 4,052,509 published a total of
29 compounds with the following pyridone-like formula (I): ##STR1##
wherein A is an aromatic group. These compounds have good
anti-inflammatory and analgesic activities and can reduce serum
levels of uric acid and glucose. One compound in particular,
5-methyl-1-phenyl-2(1H)-pyridone, has the best activity and low
toxicity.
[0007] U.S. Pat. No. 5,310,562 reported anti-fibrotic activity for
5-methyl-1-phenyl-2(1H)-pyridone (PIRFENIDONE, PFD). U.S. Pat. Nos.
5,518,729 and 5,716,632 described the anti-fibrotic activities of
additional 44 compounds of either N-substituted 2(1H)-pyridone (I)
or N-substituted 3(1H)-pyridone.
[0008] The efficacy of anti-fibrotic activity of
5-methyl-1-phenyl-2(1H)-pyridone (PIRFENIDONE, PFD) has been
further demonstrated in various animal models and human clinical
trials (Shimizu et al., Pirfenidone prevents collagen accumulation
in the remnant kidney in rats with partial nephrectomy. Kidney Int.
52(Suppl 63):S239-243 (1997); Raghu et al., Treatment of idiopathic
pulmonary fibrosis with a new antifibrotic agent, pirfenidone. Am.
J. Respir. Crit. Care Med. 159:1061-1069 (1999)). These studies
indicated that PIRFENIDONE not only prevents but also reverses the
accumulation of excess extracellular matrix. The pharmacological
mechanism of PIRFENIDONE has not been fully understood yet, but
data to date indicate that PIRFENIDONE is an effective compound to
down-regulate cytokines (including TGF-.beta.), and decrease the
activity of fibroblasts through regulating multiple factors.
[0009] A Chinese patent ZL02114190.8 described the identification
and synthesis of total 38 new 5-methyl-1-(substituted
phenyl)-2(1H)-pyridone compounds, having the following general
structural formula (II): ##STR2##
[0010] U.S. Pat. No. 5,716,632 listed 6 structural formulas of
5-methyl-1-(substituted phenyl)-2(1H)-pyridone originally described
by Gadekar in U.S. Pat. No. 3,974,281. For these "substituted
phenyl" compounds, Gadekar established a structure-activity
relationship that teaches non-substituted phenyl as the compound
with the best biological activities. However, it has been reported
that a 5-methyl-1-(substituted phenyl)-2(1H)-pyridone,
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone, displayed certain
biological activities in vitro (J. Cent. South Univ. Med. Sci.
29:139 (2004)). Thus, it is of interest to determine whether
substituted phenyl compounds having structural formula II would
have any desirable anti-fibrotic activity.
SUMMARY OF THE INVENTION
[0011] The present invention discloses a composition for treating
fibrotic diseases comprising novel compounds in the
5-methyl-1-(substituted phenyl)-2(1H)-pyridone family. The efficacy
of a representative substituted phenyl pyridone,
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone (AKF-PD), was
demonstrated in various animal models of fibrosis diseases. AKF-PD
was synthesized according to a process similar to the one described
in Chinese patent ZL02114190.8, the disclosure of which is
incorporated herein by reference. As compared with PIRFENIDONE
(PFD), a leading experimental drug in the field, AKF-PD has better
anti-fibrotic activities but with much less toxicity. The results
presented herein demonstrate that 5-methyl-1-(substituted
phenyl)-2(1H)-pyridones can be used as more potent anti-fibrotic
drugs for organ or tissue fibrosis with much less toxic effect.
[0012] In one embodiment, the present invention provides a
composition comprising one or more 5-methyl-1-(substituted
phenyl)-2(1H)-pyridone in an amount effective for treating organ or
tissue fibrosis, said 5-methyl-1-(substituted
phenyl)-2(1H)-pyridone having a general structural formula of:
##STR3## wherein n=1 or 2; R is selected from the group consisting
of F, Cl, Br, I, nitro, C.sub.1-C.sub.6 straight-chain alkyl group,
C.sub.3-C.sub.6 branched-chain alkyl group, C.sub.1-C.sub.6
straight-chain alkoxy group, C.sub.3-C.sub.6 branched-chain alkoxy
group, and halogenated C.sub.1-C.sub.6 alkyl group; and when n=2,
not both R are nitro.
[0013] The present invention also provides a group of novel
compounds of 5-methyl-1-(substituted phenyl)-2(1H)-pyridones.
[0014] The present invention also provides methods of using a
composition comprising one or more 5-methyl-1-(substituted
phenyl)-2(1H)-pyridone as disclosed herein for treating organ or
tissue fibrosis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] These and other features and advantages of this invention
will be evident from the following detailed description of
preferred embodiments when read in conjunction with the
accompanying drawings in which:
[0016] FIG. 1 shows the inhibition of TGF-.beta. production in rat
UUO model (Unilateral Ureteral Obstruction) by
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone (AKF-PD). A: Control,
sham operation; B: Disease model, UUO; C: Disease model+AKF-PD.
Brown or dark color indicate TGF-.beta. positive cells.
[0017] FIG. 2 shows the inhibition of type I & III collagen
accumulation in rat UUO model (Unilateral Ureteral Obstruction) by
AKF-PD. Section A-C: type I collagen; section D-F: type III
collagen. A and D: control rat with sham operation; B and E:
disease model with UUO; C and F, disease model+AKF-PD.
[0018] FIG. 3 shows the suppression of Schistosome-induced liver
fibrotic nodule by AKF-PD. A: Control rat; B: Fibrotic rat
(Schistosoma-induced liver fibrosis); C: Fibrotic rat+Pyquiton; D:
Fibrotic rat+Interferon-.gamma.; E: Fibrotic rat+AKF-PD. Brown or
dark color indicate fibrotic nodule.
[0019] FIG. 4 shows reduction of type I collagen accumulation in
Schistosome-induced liver fibrotic nodule by AKF-PD. A: Control
rat; B: Fibrotic rat (Schistosoma-induced liver fibrosis); C:
Fibrotic rat+Pyquiton; D: Fibrotic rat+Interferon-.gamma.; E:
Fibrotic rat+AKF-PD. Brown or dark color indicate collagen I
staining.
DETAILED DESCRIPTION OF THE INVENTION
[0020] In the present invention, the term "anti-organ or tissue
fibrosis" means preventing fibrosis in organs or tissues, slowing
or stopping fibrosis process in organs or tissues, and/or reversing
the fibrotic lesion in organs or tissues.
[0021] It was shown previously that
5-methyl-1-phenyl-2(1H)-pyridone (PIRFENIDONE) with non-substituted
phenyl is a compound with desirable anti-fibrotic activities, which
is consistent with Gadekar's structure-activity relationship:
non-substituted phenyl is better than substituted phenyl. Thus, it
is unexpected to find 5-methyl-1-(substituted
phenyl)-2(1H)-pyridones (structural formula II) exhibiting more
potent anti-fibrotic activities and much less toxicity than
5-methyl-1-phenyl-2(1H)-pyridone (PIRFENIDONE) as shown herein.
[0022] The present invention discloses a new structure-activity
relationship: 5-methyl-1-(substituted phenyl)-2(1H)-pyridones are
better than 5-methyl-1-phenyl-2(1H)-pyridone. In structural formula
II, when n=1, the following compounds all have activities
inhibiting fibroblast: 1-(3'-bromophenyl)-5-methyl-2(1H)-pyridone,
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone,
1-(3'-iodophenyl)-5-methyl-2(1H)-pyridone,
5-methyl-1-(3'-methylphenyl)-2(1H)-pyridone. A side-by-side
comparison further indicates that the inhibitory activity to
fibroblasts is: F>Br>Cl>H.
[0023] A representative example of 5-methyl-1-(substituted
phenyl)-2(1H)-pyridone is
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone. It was determined that
the anti-fibrotic activities of
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone are enhanced compared
to PIRFENIDONE. Moreover, the toxicity of 5-methyl-1-(substituted
phenyl)-2(1H)-pyridone is drastically decreased. As presented
herein, the LD.sub.50 for
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone is only 30% percent of
that of PIRFENIDONE. It is important to note that as all fibrotic
diseases are chronic illness, it is expected that any reparation or
prevention is going to be a lengthy process, which warrants a
long-term pharmacological intervention. Therefore, just like any
long-term drug use, it is very desirable to have anti-fibrotic
drugs with low toxicity.
[0024] Other examples of 5-methyl-1-(substituted
phenyl)-2(1H)-pyridone (structural formula II) include, but are not
limited to, the following compounds:
[0025] When n=1 and R.dbd.Br, the compounds can be
1-(2'-bromophenyl)-5-methyl-2(1H)-pyridone,
1-(3'-bromophenyl)-5-methyl-2(1H)-pyridone, or
1-(4'-bromophenyl)-5-methyl-2(1H)-pyridone.
[0026] When n=1 and R.dbd.F, the compounds can be
1-(2'-fluorophenyl)-5-methyl-2(1H)-pyridone,
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone, or
1-(4'-fluorophenyl)-5-methyl-2(1H)-pyridone.
[0027] When n=1 and R.dbd.I, the compounds can be
1-(2'-iodophenyl)-5-methyl-2(1H)-pyridone,
1-(3'-iodophenyl)-5-methyl-2(1H)-pyridone, or
1-(4'-iodophenyl)-5-methyl-2(1H)-pyridone.
[0028] When n=2 and R.dbd.F, Br, or Cl, the compounds can be
1-(2',3'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(3',5'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(2',3'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(3',5'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',3'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-difluorophenyl)-5-methyl-2(1H)-pyridone, or
1-(3',5'-difluorophenyl)-5-methyl-2(1H)-pyridone.
[0029] When n=1 or 2 and R=trifluoromethyl, the compounds can be
5-methyl-1-(2'-trifluoromethylphenyl)-2(1H)-pyridone,
5-methyl-1-(4'-trifluoromethylphenyl)-2(1H)-pyridone,
1-(2',3'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone, or
1-(3',5'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone.
[0030] When n=1 or 2 and R=methyl, the compounds can be
5-methyl-1-(2'-methylphenyl)-2(1H)-pyridone,
5-methyl-1-(3'-methylphenyl)-2(1H)-pyridone,
1-(2',3'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-dimethylphenyl)-5-methyl-2(1H)-pyridone, or
1-(3',5'-dimethylphenyl)-5-methyl-2(1H)-pyridone.
[0031] When n=1 or 2 and R=methoxy, the compounds can be
1-(2'-methoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(3'-methoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(2',3'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone, or
1-(3',5'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone.
[0032] The present invention provides a composition comprising one
or more 5-methyl-1-(substituted phenyl)-2(1H)-pyridone in an amount
effective for treating organ or tissue fibrosis, said
5-methyl-1-(substituted phenyl)-2(1H)-pyridone having a general
structural formula of: ##STR4## wherein n=1 or 2; R is selected
from the group consisting of F, Cl, Br, I, nitro, C.sub.1-C.sub.6
straight-chain alkyl group, C.sub.3-C.sub.6 branched-chain alkyl
group, C.sub.1-C.sub.6 straight-chain alkoxy group, C.sub.3-C.sub.6
branched-chain alkoxy group, and halogenated C.sub.1-C.sub.6 alkyl
group; and when n=2, not both R are nitro.
[0033] Examples of 5-methyl-1-(substituted phenyl)-2(1H)-pyridones
in the above composition include, but are not limited to,
1-(2'-bromophenyl)-5-methyl-2(1H)-pyridone,
1-(3'-bromophenyl)-5-methyl-2(1H)-pyridone,
1-(4'-bromophenyl)-5-methyl-2(1H)-pyridone,
1-(3'-chlorophenyl)-5-methyl-2(1H)-pyridone,
1-(2'-fluorophenyl)-5-methyl-2(1H)-pyridone,
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone,
1-(4'-fluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2'-iodophenyl)-5-methyl-2(1H)-pyridone,
1-(3'-iodophenyl)-5-methyl-2(1H)-pyridone,
1-(4'-iodophenyl)-5-methyl-2(1H)-pyridone,
1-(2',3'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(3',5'-dibromophenyl)-5-methyl-2(1H)-pyridone,
1-(2',3'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(3',5'-dichlorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',3'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(3',5'-difluorophenyl)-5-methyl-2(1H)-pyridone,
5-methyl-1-(2'-trifluoromethylphenyl)-2(1H)-pyridone,
5-methyl-1-(4'-trifluoromethylphenyl)-2(1H)-pyridone,
1-(2',3'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone, or
1-(3',5'-bis-trifluoromethylphenyl)-5-methyl-2(1H)-pyridone.
5-methyl-1-(2'-methylphenyl)-2(1H)-pyridone,
1-(3'-methylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',3'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(3',5'-dimethylphenyl)-5-methyl-2(1H)-pyridone,
1-(2'-methoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(3'-methoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(2',3'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone, and
1-(3',5'-dimethoxyphenyl)-5-methyl-2(1H)-pyridone.
[0034] The present invention also provides a compound of
5-methyl-1-(substituted phenyl)-2(1H)-pyridone. Examples of such
compounds include, but are not limited to,
1-(2'-fluorophenyl)-5-methyl-2(1H)-pyridone,
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone,
1-(4'-fluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2'-iodophenyl)-5-methyl-2(1H)-pyridone,
1-(3'-iodophenyl)-5-methyl-2(1H)-pyridone,
1-(4'-iodophenyl)-5-methyl-2(1H)-pyridone,
1-(2',3-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',4'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',5'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(2',6'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(3',4'-difluorophenyl)-5-methyl-2(1H)-pyridone,
1-(3',5'-difluorophenyl)-5-methyl-2(1H)-pyridone and
1-(3',4'-dichlorophenyl)-5-methyl-2(1H)-pyridone.
[0035] The present invention also provides a pharmaceutical
composition comprising the composition described above and a
pharmaceutically acceptable carrier. The pharmaceutical composition
can be formulated as solution, tablet, capsule, suppository,
inhaler, suspension, gel, cream, or ointment.
[0036] The present invention also provides a method of treating
organ or tissue fibrosis, comprising the step of administrating a
composition comprising one or more compound as described above. The
method can be used to treat organ or tissue fibrosis such as
glomerulus sclerosis, renal interstitial fibrosis, liver fibrosis,
pulmonary fibrosis, peridoneal fibrosis, myocardiac fibrosis,
fibrosis of skin, post-surgical adhesion, benign prostatic
hypertrophy, musculoskeletal fibrosis, scleroderma, Alzheimer's
disease, fibrotic vascular disease, and glaucoma. Examples of
amounts effective for treating organ or tissue fibrosis include a
daily dosage of about 25 mg to about 6,000 mg, a daily dosage of
about 50 mg to about 2000 mg, or a daily dosage of about 100 mg to
about 1000 mg. The composition described above can be administered
by oral administration, parenteral administration, nasal
administration, rectal administration, vaginal administration,
ophthalmic application, or topical application.
[0037] In one embodiment, the above method comprises administrating
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone to a subject in need of
such treatment. Subject treated with
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone will experience less
toxicity than treatment with 5-methyl-1-phenyl-2(1H)-pyridone.
[0038] The invention being generally described will be more readily
understood by reference to the following examples which are
included merely for purposes of illustration of certain aspects and
embodiments of the present invention, and are not intended to limit
the invention.
[0039] In the following examples, myocardiac fibroblasts, skin scar
forming fibroblast and human peritoneal mesothelial cells are
primary cultures prepared according to common procedure. Other
cells are commercially purchased.
1-(3-fluorophenyl)-5-methyl-2(1H)-pyridone (AKF-PD) is prepared as
a suspension in 5% carboxymethylcellulose.
EXAMPLE 1
Suppression of Mouse Kidney Fibroblast Proliferation by
1-(3'-Fluorophenyl)-5-Methyl-2(1H)-Pyridone and PIRFENIDONE
[0040] Cell proliferation was measured by MTT assay. DMEM with 10%
fetal serum was used as cell culture medium. The cells were
prepared in suspension (1.times.10.sup.5/ml), and 100 .mu.L of the
suspension was transferred to each well of a 96 wells plate. Once
the cells were attached to the plastic, the culture was changed to
serum free medium and continued for another 24 hours. The serum
free medium was aspirated, and various concentrations of
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone (AKF-PD)
1-(3'-bromophenyl)-5-methyl-2(1H)-pyridone (AKF-BR) or PIRFENIDONE
(PFD) in 10% serum medium were added into each well with 5
replicates for each concentration. The cells were stained with MTT
(10 .mu.L per well) at 24, 48, and 72 hours post drug treatment.
After 4 hrs of incubation, the medium with MTT was aspirated from
each well. One hundred .mu.L MTT solvent was added to each well for
15 min. The dissolved MTT was then measured with a plate reader at
570 nm.
[0041] The results are shown in Table 1. Both
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone (AKF-PD) and
1-(3'-bromophenyl)-5-methyl-2(1H)-pyridone (AKF-BR) were capable of
inhibiting the proliferation of kidney fibroblast. The inhibitory
effect was AKF-PD>AKF-BR>pirfenidone. AKF-PD showed a
significantly stronger inhibitory effect than pirfenidone.
TABLE-US-00001 TABLE 1 Effects of AKF-PD, AKF-BR and PFD on Kidney
Fibroblast Optical Density at 570 nm Group 24 h 48 h 72 h Control
0.5978 .+-. 0.0143 0.5994 .+-. 0.0124 0.6338 .+-. 0.0095 AKF-PD 100
.mu.g/ml 0.5907 .+-. 0.0199 0.5850 .+-. 0.0134 0.5080 .+-. 0.0145*
AKF-PD 500 .mu.g/ml 0.5799 .+-. 0.1086 0.5185 .+-. 0.0331 0.4314
.+-. 0.0264*# AKF-PD 1000 .mu.g/ml 0.5638 .+-. 0.0142* 0.4298 .+-.
0.0258*# 0.3511 .+-. 0.0215*# AKF-BR 100 .mu.g/ml 0.5937 .+-.
0.0307 0.5811 .+-. 0.0161 0.5363 .+-. 0.0158* AKF-BR 500 .mu.g/ml
0.5895 .+-. 0.0253 0.5418 .+-. 0.0221* 0.4731 .+-. 0.0249* AKF-BR
1000 .mu.g/ml 0.5723 .+-. 0.0254 0.5192 .+-. 0.0178* 0.4161 .+-.
0.0249* PFD 1000 .mu.g/ml 0.5911 .+-. 0.1002 0.5844 .+-. 0.0171
0.5264 .+-. 0.1530 PFD 500 .mu.g/ml 0.5877 .+-. 0.1204 0.5450 .+-.
0.0196* 0.4798 .+-. 0.2355* PFD 100 .mu.g/ml 0.5798 .+-. 0.0149
0.5272 .+-. 0.0229* 0.4269 .+-. 0.0302* *p < 0.05 vs control; #p
< 0.05 vs PFD
EXAMPLE 2
Suppression of Rat Myocardiac Fibroblasts Proliferation by
1-(3'-Fluorophenyl)-5-Methyl-2(1H)-Pyridone and PIRFENIDONE
[0042] Cell proliferation was measured by MTT assay. DMEM with 10%
fetal serum was used as cell culture medium. The cells were
prepared in suspension (1.times.10.sup.5/ml), and 100 .mu.L of the
suspension was transferred to each well of a 96 wells plate. Once
the cells were attached to the plastic, the culture was changed to
serum free medium and incubated for another 24 hours. Then, the
serum free medium was aspirated, and various concentrations of
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone (AKF-PD) or PIRFENIDONE
(PFD) in 10% serum medium were added into each well with 5
replicates for each concentration. The cells were stained with MTT
(10 .mu.L per well) at 12, 24, or 48 hours post drug treatment.
After 4 hrs of incubation, the medium with MTT was aspirated from
each well. One hundred .mu.L MTT solvent was added to each well for
15 min, and the dissolved MTT was measured with a plate reader at
570 nm.
[0043] The results are shown in Table 2. At concentrations of 100
.mu.g/ml, 500 .mu.g/ml, 1000 .mu.g/ml and 2500 .mu.g/ml, both
AKF-PD and PIRFENIDONE can suppress proliferation of rat myocardiac
fibroblasts after 24 hours treatment; however, at 1000 .mu.g/ml and
2500 .mu.g/ml levels, AKF-PD was more potent than PIRFENIDONE. At
the same concentration ranges, both AKF-PD and PIRFENIDONE showed
suppressive effects on cell proliferation after 48 hours, but
AKF-PD was more potent at 100 .mu.g/ml, 500 .mu.g/ml, and 1000
.mu.g/ml. In conclusion, AKF-PD is a more potent anti-proliferative
agent than PIRFENIDONE on rat myocardiac fibroblasts.
TABLE-US-00002 TABLE 2 Effects of AKF-PD and PFD on Rat Myocardiac
Fibroblasts Optical Density at 570 nm Group 12 h 24 h 48 h control
0.330 .+-. 0.002 0.445 .+-. 0.016 0.684 .+-. 0.008 AKF-PD 0.328
.+-. 0.010 0.426 .+-. 0.006* 0.620 .+-. 0.018*** 100 .mu.g/ml
AKF-PD 0.326 .+-. 0.003 0.408 .+-. 0.009** 0.580 .+-. 0.014*** 500
.mu.g/ml AKF-PD 0.332 .+-. 0.006 0.392 .+-. 0.008** 0.538 .+-.
0.009*** 1000 .mu.g/ml AKF-PD 0.325 .+-. 0.008 0.377 .+-. 0.013***
0.514 .+-. 0.005*** 2500 .mu.g/ml PFD 0.330 .+-. 0.014 0.429 .+-.
0.009* 0.654 .+-. 0.007*.sup.+ 100 .mu.g/ml PFD 0.329 .+-. 0.013
0.411 .+-. 0.006* 0.612 .+-. 0.014***.sup.++ 500 .mu.g/ml PFD 0.331
.+-. 0.009 0.403 .+-. 0.010**.sup.+ 0.597 .+-. 0.013***.sup.+++
1000 .mu.g/ml PFD 0.329 .+-. 0.008 0.392 .+-. 0.009**.sup.+ 0.566
.+-. 0.027** 2500 .mu.g/ml *p < 0.05 vs control; .sup.+p <
0.05 vs AKF-PD; **p < 0.01 vs control; .sup.++p < 0.01 vs
AKF-PD; ***p < 0.001 vs control; .sup.+++p < 0.001 vs
AKF-PD;
EXAMPLE 3
Suppression of Human Stellate Cell Proliferation by
1-(3'-Fluorophenyl)-5-Methyl-2(1H)-Pyridone and PIRFENIDONE
[0044] Cell proliferation was measured by MTT assay. DMEM with 10%
fetal serum was used as cell culture medium. The cells were
prepared in suspension (1.times.10.sup.5/ml), and 100 .mu.L of the
suspension was transferred to each well of a 96 wells plate. Once
the cells were attached to the plastic, the culture was changed to
serum free medium and incubated for another 24 hours. Then, the
serum free medium was aspirated, and various concentrations of
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone (AKF-PD) or PIRFENIDONE
(PFD) in 10% serum medium were added into each well with 5
replicates for each concentration. The cells were stained with MTT
(10 .mu.L per well) at 12, 24, or 48 hours post drug treatment.
After 4 hrs of incubation, the medium with MTT was aspirated from
each well. One hundred .mu.L MTT solvent was added to each well for
15 min, and the dissolved MTT was then measured with a plate reader
at 570 nm.
[0045] The results are shown in Table 3. At 500 .mu.g/ml, 1000
.mu.g/ml, and 2500 .mu.g/ml, both AKF-PD and PIRFENIDONE can
suppress proliferation of human stellate cells beginning from 12
hours post drug treatment. At the 24 hours time point, 1000
.mu.g/ml and 2500 .mu.g/ml of AKF-PD was more suppressive than
PIRFENIDONE. At 48 hours, AKF-PD was more suppressive than
PIRFENIDONE at concentrations of 500 .mu.g/ml, 1000 .mu.g/ml, and
2500 .mu.g/ml. In conclusion, AKF-PD is a more potent
anti-proliferative agent than PIRFENIDONE on human stellate cells.
TABLE-US-00003 TABLE 3 Effects of AKF-PD and PFD on Human Stellate
Cells Optical Density at 570 nm Group 12 h 24 h 48 h Control 0.207
.+-. 0.001 0.370 .+-. 0.002 0.455 .+-. 0.002 AKF-PD 100 .mu.g/ml
0.202 .+-. 0.001 0.366 .+-. 0.002 0.442 .+-. 0.006 AKF-PD 500
.mu.g/ml 0.202 .+-. 0.001* 0.341 .+-. 0.003** 0.406 .+-. 0.002***
AKF-PD 1000 .mu.g/ml 0.198 .+-. 0.001** 0.312 .+-. 0.003*** 0.385
.+-. 0.004*** AKF-PD 2500 .mu.g/ml 0.195 .+-. 0.002** 0.273 .+-.
0.005*** 0.246 .+-. 0.001*** PFD 100 .mu.g/ml 0.206 .+-. 0.003
0.371 .+-. 0.001 0.447 .+-. 0.003 PFD 500 .mu.g/ml 0.202 .+-.
0.001* 0.345 .+-. 0.002** 0.413 .+-. 0.001***.sup.++ PFD 1000
.mu.g/ml 0.201 .+-. 0.001* 0.330 .+-. 0.001***.sup.++ 0.402 .+-.
0.001***.sup.++ PFD 2500 .mu.g/ml 0.198 .+-. 0.001** 0.278 .+-.
0.001***.sup.+ 0.306 .+-. 0.002***.sup.+++ *p < 0.05 vs control;
.sup.+p < 0.05 vs AKF-PD; **p < 0.01 vs control; .sup.++p
< 0.01 vs AKF-PD; ***p < 0.001 vs control; .sup.+++p <
0.001 vs AKF-PD;
EXAMPLE 4
Suppression of Rat Pulmonary Fibroblast Proliferation by
1-(3'-Fluorophenyl)-5-Methyl-2(1H)-Pyridone,
1-(3'-bromophenyl)-5-Methyl-2(1H)-Pyridone and PIRFENIDONE
[0046] Cell proliferation was measured by MTT assay. DMEM with 10%
fetal serum was used as cell culture medium. The cells were
prepared in suspension (1.times.10.sup.5/ml), and 100 .mu.L of the
suspension was transferred to each well of a 96 wells plate. Once
the cells were attached to the plastic, the culture was changed to
serum free medium and incubated for another 24 hours. Then, the
serum free medium was aspirated, and various concentrations of
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone (AKF-PD) or
1-(3'-bromophenyl)-5-Methyl-2(1H)-Pyridone or PIRFENIDONE (PFD) in
10% serum medium were added into each well with 5 replicates for
each concentration. The cells were stained with MTT (10 .mu.L per
well) at 24, or 48 hours post drug treatment. After 4 hrs of
incubation, the medium with MTT was aspirated from each well. One
hundred .mu.L MTT solvent was added to each well for 15 min, and
the dissolved MTT was measured with a plate reader at 570 nm.
[0047] The results are shown in Table 4. Both
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone (AKF-PD) and
1-(3'-bromophenyl)-5-methyl-2(1H)-pyridone (AKF-BR) could suppress
proliferation of rat pulmonary fibroblasts. The inhibitory effect
among the test compounds was AKF-PD>AKF-BR>pirfenidone.
AKF-PD has a significant stronger inhibitory effect than
pirfenidone does. TABLE-US-00004 TABLE 4 Effects of AKF-PD, AKF-BR
and PFD on Rat Pulmonary Fibroblasts Optical Density at 570 nm
Group 24 h 48 h Control 0.1713 .+-. 0.0226 0.1754 .+-. 0.0167
AKF-PD 100 .mu.g/ml 0.1467 .+-. 0.0138* 0.1369 .+-. 0.0115*.sup.#
AKF-PD 500 .mu.g/ml 0.1258 .+-. 0.0119* 0.1214 .+-. 0.0234*.sup.#
AKF-PD 1000 .mu.g/ml 0.1130 .+-. 0.0163* 0.1119 .+-. 0.0285*.sup.#
AKF-BR 100 .mu.g/ml 0.1654 .+-. 0.0143 0.1475 .+-. 0.0211* AKF-BR
500 .mu.g/ml 0.1342 .+-. 0.0237* 0.1292 .+-. 0.0178*.sup.# AKF-BR
1000 .mu.g/m1 0.1204 .+-. 0.0176* 0.1201 .+-. 0.0342*.sup.# PFD 100
.mu.g/ml 0.2023 .+-. 0.0169 0.1864 .+-. 0.0530 PFD 500 .mu.g/ml
0.1887 .+-. 0.0130 0.1459 .+-. 0.0255* PFD 1000 .mu.g/m1 0.1797
.+-. 0.0166 0.1269 .+-. 0.0302* *p < 0.05 vs control; .sup.#p
< 0.05 vs PFD
EXAMPLE 5
Suppression of Human Skin Scar Forming Fibroblast Proliferation by
1-(3'-Fluorophenyl)-5-Methyl-2(1H)-Pyridone and PIRFENIDONE
[0048] Cell proliferation was measured by MTT assay. DMEM with 10%
fetal serum was used as cell culture medium. The cells were
prepared in suspension (1.times.10.sup.5/ml), and 100 .mu.L of the
suspension was transferred to each well of a 96 wells plate. Once
the cells were attached to the plastic, the culture was changed to
serum free medium and incubated for another 24 hours. Then, the
serum free medium was aspirated, and various concentrations of
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone (AKF-PD) or PIRFENIDONE
(PFD) in 10% serum medium were added into each well with 5
replicates for each concentration. The cells were stained with MTT
(10 .mu.L per well) at 12, 24, or 48 hours post drug treatment.
After 4 hrs of incubation, the medium with MTT was aspirated from
each well. One hundred .mu.L MTT solvent was added to each well for
15 min, and the dissolved MTT was measured with a plate reader at
570 nm.
[0049] The results are shown in Table 5. After drug treatment for
24 hrs at concentrations of 100 .mu.g/ml, 500 .mu.g/ml, 1000
.mu.g/ml, and 2500 .mu.g/ml, both AKF-PD and PIRFENIDONE were
capable of inhibiting the growth of human skin fibroblasts;
however, AKF-PD was more potent than PIRFENIDONE at concentrations
of 500 .mu.g/ml, 1000 .mu.g/ml, and 2500 .mu.g/ml. After 48 hrs of
treatment, 500 .mu.g/ml or 1000 .mu.g/ml of AKF-PD showed more
inhibition than similar concentrations of PIRFEIDONE. In
conclusion, AKF-PD is a more potent anti-proliferative agent than
PIRFENIDONE on human skin fibroblasts. TABLE-US-00005 TABLE 5
Effects of AKF-PD and PFD on Human Skin Scar Forming Fibroblasts
Optical Density at 570 nm Group 12 h 24 h 48 h Control 0.195 .+-.
0.008 0.263 .+-. 0.005 0.381 .+-. 0.001 AKF-PD 0.192 .+-. 0.010
0.245 .+-. 0.002* 0.366 .+-. 0.006* 100 .mu.g/ml AKF-PD 0.192 .+-.
0.006 0.238 .+-. 0.004* 0.345 .+-. 0.007* 500 .mu.g/ml AKF-PD 0.192
.+-. 0.009 0.221 .+-. 0.004** 0.323 .+-. 0.009*** 1000 .mu.g/ml
AKF-PD 0.190 .+-. 0.002 0.198 .+-. 0.008*** 0.267 .+-. 0.001***
2500 .mu.g/ml PFD 0.194 .+-. 0.004 0.250 .+-. 0.003* 0.366 .+-.
0.006* 100 .mu.g/ml PFD 0.191 .+-. 0.008 0.245 .+-. 0.004*.sup.+
0.350 .+-. 0.003***.sup.++ 500 .mu.g/ml PFD 0.190 .+-. 0.008 0.330
.+-. 0.001*.sup.++ 0.328 .+-. 0.004***.sup.++ 1000 .mu.g/ml PFD
0.193 .+-. 0.004 0.278 .+-. 0.001***.sup.+++ 0.264 .+-. 0.005***
2500 .mu.g/ml *p < 0.05 vs control; .sup.+p < 0.05 vs AKF-PD;
**p < 0.01 vs control; .sup.++p < 0.01 vs AKF-PD; ***p <
0.001 vs control; .sup.+++p < 0.001 vs AKF-PD;
EXAMPLE 6
Suppression of Human Peritoneal Mesothelial Cell Proliferation by
1-(3'-Fluorophenyl)-5-Methyl-2(1H)-Pyridone and PIRFENIDONE
[0050] Cell proliferation was measured by MTT assay. DMEM with 10%
fetal serum was used as cell culture medium. The cells were
prepared in suspension (1.times.10.sup.5/ml), and 100 .mu.L of the
suspension was transferred to each well of a 96 wells plate. Once
the cells were attached to the plastic, the culture was changed to
serum free medium and incubated for another 24 hours. Then, the
serum free medium was aspirated, and various concentrations of
1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone (AKF-PD) or PIRFENIDONE
(PFD) in 10% serum medium were added into each well with 5
replicates for each concentration. The cells were stained with MTT
(10 .mu.L per well) at 12, 22, or 48 hours post drug treatment.
After 4 hrs of incubation, the medium with MTT was aspirated from
each well. One hundred .mu.L MTT solvent was added to each well for
15 min, and the dissolved MTT was measured with a plate reader at
570 nm.
[0051] The results are shown in Table 6. After drug treatment for
24 hrs at concentrations of 500 .mu.g/ml, 1000 .mu.g/ml, and 2500
.mu.g/ml, both AKF-PD and pirfenidone were capable of inhibiting
the growth of human peritoneal mesothelial cells; however, AKF-PD
was more potent than PIRFENIDONE at concentrations of 1000 .mu.g/ml
and 2500 .mu.g/ml. For 48 hrs of treatment, 1000 .mu.g/ml of AKF-PD
showed more inhibition than similar concentration of PIRFEIDONE. In
conclusion, AKF-PD is a more potent anti-proliferative agent than
PIRFENIDONE on human peritoneal mesothelial cells. TABLE-US-00006
TABLE 6 Effects of AKF-PD and PFD on Human Peritoneal Mesothelial
Cells Optical Density at 570 nm Group 12 h 24 h 48 h Control 0.347
.+-. 0.006 0.585 .+-. 0.002 0.814 .+-. 0.003 AKF-PD 0.344 .+-.
0.004 0.583 .+-. 0.004 0.807 .+-. 0.007 100 .mu.g/ml AKF-PD 0.344
.+-. 0.005 0.573 .+-. 0.004* 0.758 .+-. 0.010* 500 .mu.g/ml AKF-PD
0.343 .+-. 0.004 0.553 .+-. 0.004*** 0.704 .+-. 0.003*** 1000
.mu.g/ml AKF-PD 0.346 .+-. 0.005 0.502 .+-. 0.003*** 0.646 .+-.
0.006*** 2500 .mu.g/ml PFD 0.346 .+-. 0.006 0.584 .+-. 0.005 0.810
.+-. 0.006 100 .mu.g/ml PFD 0.343 .+-. 0.004 0.577 .+-. 0.003*
0.766 .+-. 0.004*** 500 .mu.g/ml PFD 0.363 .+-. 0.003 0.563 .+-.
0.003***.sup.+ 0.714 .+-. 0.002***.sup.+++ 1000 pg/ml PFD 0.345
.+-. 0.005 0.512 .+-. 0.006***.sup.+ 0.648 .+-. 0.009*** 2500 pg/ml
*p < 0.05 vs control; .sup.+p < 0.05 vs AKF-PD; **p < 0.01
vs control; .sup.++p < 0.01 vs AKF-PD; ***p < 0.001 vs
control; .sup.+++p < 0.001 vs AKF-PD;
EXAMPLE 7
Effects of 1-(3'-Fluorophenyl)-5-Methyl-2(1H)-Pyridone (AKF-PD) on
Glomerulosclerosis and Interstitial Renal Fibrosis
[0052] Rat diabetic kidney disease was induced by streptozotocin
(STZ) to test the effectiveness of 5-methyl-2(1H)-pyridone (AKF-PD)
in preventing glomerulosclerosis and renal interstitial
fibrosis.
[0053] Eight-week old male Wistar rat (180 g.about.220 g) were
randomly grouped as normal, diabetic nephrosis, diabetic
nephrosis/valsartan, or diabetic nephrosis/AKF-PD. Diabetes was
induced by a single i.p. injection of STZ 55 mg/kg. Diabetic
condition was confirmed 24 hours later by the following test
results: 13.9 mmol/L of fasting blood-glucose, 16.7 mmol/L random
blood-glucose, and positive urine-glucose. If the diabetic rat had
urine protein level higher than 30 mg/d 4 weeks later, a rat model
was established for diabetic nephrosis.
[0054] Rats in the group of diabetic nephrosis/AKF-PD were orally
fed 500 mg/kg/d of AKF-PD, and rats in the group of diabetic
nephrosis/valsartan were fed 30 mg/kg/d of valsartan. Normal saline
was fed to rats in the group of diabetic nephrosis. After 12 weeks
of drug treatment, all rats were sacrificed and their kidneys were
removed for pathological examination. The references for standard
histopathological scoring system for glomerulus and renal tubules
interstitial tissue are: Radford et al., Predicting renal outcome
in IgA nephropathy. J. Am. Soc. Nephrol. 8(2) 199-207(1997); Zhao
et al., Comparison of renoprotective effect between Angiotensin II
receptor antagonist and angiotensin-converting enzyme inhibitor on
puromycin nephropathy and their possible mechanism. Chin. J. Mult.
Organ Dis. Elderly 1(1) 36-40(2002).
[0055] The results of microscopic examination of kidney tissues are
shown in Table 7. Compared to diabetic nephrosis rats without any
treatment, the AKF-PD-treated rats showed less damage on their
glomerulus and renal tubules interstitial tissue, indicating that
AKF-PD may effectively treat glomerulus sclerosis and renal tubules
interstitial fibrosis caused by diabetic condition. TABLE-US-00007
TABLE 7 Histopathological Scores From Different Treatment Groups
Renal Tubules Number of Interstitial Tissue Groups Animals
Glomerular Score Score Diabetic 5 1.29 +- 0.18 2.20 +- 0.14
Nephrosis + AKF-PD Diabetic 8 1.36 +- 0.24 3.12 +- 0.64 Nephrosis +
Valsartan Diabetic 9 1.63 +- 0.33 3.33 +- 0.54 Nephrosis Normal Rat
5 0.21 +- 0.04 0.48 +- 0.14
EXAMPLE 8
Effects of 1-(3'-Fluorophenyl)-5-Methyl-2(1H)-Pyridone (AKF-PD) in
a Rat Model of Renal Interstitial Fibrosis
[0056] Anti-fibrotic effect of AKF-PD was tested on a SD rat model
for renal interstitial fibrosis induced by surgical ligation of
single side ureter (Unilateral Ureteral Obstruction, UUO, model).
Eight weeks old male SD rats (180 g.about.220 g) were randomly
divided into sham surgical group, disease model group, Enalapril
(10 mg/kg/d) group and AKF-PD (500 mg/kg/d) group. Under aseptic
condition, all animals in the groups of disease model, Enalapril
and AKF-PD had a surgical procedure for ligation of the left side
ureter. The animals of the sham surgical group experienced the same
surgical procedure except the ligation step. The respective drugs
were administrated by gavage to rats in the groups of Enalapril and
AKF-PD from one day prior to the procedure to 14 days post surgical
procedure. Normal saline was administrated in a similar fashion to
the rats in the groups of disease model and sham surgical. The
animals were sacrificed 14 days after surgical procedure and their
left kidney were removed for pathological (HE staining)
examination. Histological scoring for interstitial compartment was
done according to Radford's method (Radford et al., Predicting
renal outcome in IgA nephropathy. J. Am. Soc. Nephrol. 8:
199-207(1997)). As shown in Table 8, rats treated with AKF-PD
showed reduced lesion on interstitial tissues comparing to those in
the groups of disease model and Enalapril, indicating AKF-PD may be
an effective drug for interstitial renal fibrosis. TABLE-US-00008
TABLE 8 Comparison of Histological Scores for Interstitial
Compartment Sham Disease Group surgical Model Enalapril AKF-PD
Number of rats 10 9 9 8 (n) Score 1.33 .+-. 0.58 10.24 .+-. 0.99
9.21 .+-. 0.64 6.43 .+-. 1.28
EXAMPLE 9
Inhibition of TGF-.beta. Production And Type I & III Collagen
Accumulation In Rat UUO Model (Unilateral Ureteral Obstruction) by
1-(3'-Fluorophenyl)-5-Methyl-2(1H)-Pyridone (AKF-PD)
[0057] Similar to the example 8, Kidney fibrosis was induced by
Unilateral Ureteral Obstruction. And the animal experiments were
performed to investigate whether AKF-PD has any effect on
TGF-.beta. production and collagen accumulation. These are two
known factors for fibrotic lesion.
[0058] As shown by FIG. 1, sections A-C were immunohistochemically
stained with specific antibody against TGF-.beta. with brown color
indicating TGF-.beta. protein expression in kidney cells. All
sections were also counterstained lightly with hematoxylin for
viewing non-TGF-.beta. stained cells. The result indicates that the
administration of AKF-PD to the fibrotic rats have significantly
reduced TGF-.beta. production.
[0059] Also shown by FIG. 2, type I and III collagen were analyzed
by immunohistochemistry. The typical fibrotic fiber was viewed as
brown color. There are a robotic accumulation of both type I (B)
and type III (E) collagen on the fibrotic kidney of UUO model.
However, treatment of the diseased rat with AKF-PD significantly
blocks type I and III collagen accumulation or production. Reduced
collagen level can be interpreted as the reduced ECM.
[0060] The decreased TGF-.beta. production and reduced accumulation
of ECM indicates a possible therapeutic effect of AKF-PD for kidney
fibrotic condition.
EXAMPLE 10
Effects of 1-(3'-Fluorophenyl)-5-Methyl-2(1H)-Pyridone (AKF-PD) on
Liver Fibrosis
[0061] Kun Ming (KM) mice were infected with Schistosoma miracidium
to induce schistosomial liver fibrosis. Four to six weeks old male
Kun Ming (KM) mice (18-22 g) were randomly grouped into healthy,
infected, infected/Pyquiton, infected/.gamma.-interferon, and
infected/AKF-PD groups. Ten Schistosoma miracidium were placed on
shaved abdominal skin of each mouse for infection. Eight weeks
post-infection, mice in the groups of infected/Pyquiton,
infected/.gamma.-interferon, and infected/AKF-PD were disinfected
by treating with 650 mg/kg Pyquiton for 4 days. Five hundred mg/kg
AKF-PD were given daily by gavage to the disinfected mice of
infected/AKF-PD group. The disinfected mice were given (i.m.)
50,000 unit .gamma.-interferon daily. The disinfected mice in the
infected/Pyquiton and infected groups were orally given normal
saline once a day in the same way as administration of drug
treatment. Drug or normal saline treatment was continued for 8
weeks. All mice were sacrificed one week after discontinue of drug
treatment, and the left lobe of liver from each mouse was taken for
pathological examination.
[0062] Examination of HE-stained slices of liver tissue were
carried out as follows. In general, after 16 weeks of Schistosoma
miracidia infection, the area of schistosomo egg granuloma would
directly correlate to the severity of liver fibrosis. Therefore,
the area of schistosomo egg granuloma was measured using a
high-resolution, color pathology graphic analyzer (HPIAS-1000). The
sum of area of 5 granuloma with abundant eggs and the sum of area
of 5 granuloma with few eggs were measured for each slice. As shown
in Tables 9 and 10, AKF-PD-treated animals had smaller area
(.mu.m.sup.2) of schistosomo egg granuloma than those in either
no-treatment (infected only) or Pyquiton-treated group.
TABLE-US-00009 TABLE 9 Comparison of Schistosomo Egg Granuloma Area
Number Group of of Mean Granuloma animal/treatment animal
(.mu.m.sup.2) S.D. With Infected/.gamma.-interferon 9 83706.79
22943.48 Abundant Infected/AKF-PD. 10 80155.11 25419.82 Eggs
Infected only 10 111604.59 30115.49 Infected/Pyquiton 11 125823.35
31708.85 With Infected/.gamma.-interferon 9 32407.14 10078.30 Few
Infected/AKF-PD. 10 30266.68 11069.89 Eggs Infected only 10
41116.13 11246.94 Infected/Pyquiton 11 45418.59 18001.53
[0063] TABLE-US-00010 TABLE 10 P values for Groups in Table 9 With
abundant With few Groups eggs eggs Infected only vs
infected/.gamma.- 0.038 0.095 interferon,
infected/.gamma.-interferon, vs 0.004 0.058 infected/Pyquiton
infected/Pyquiton vs infected/AKF- 0.002 0.032 PD. Infected only vs
infected/Pyquiton 0.306 0.516 Infected only vs infected/AKF-PD.
0.021 0.043 infected/.gamma.-interferon vs 0.754 0.666
infected/AKF-PD
[0064] Some representative immunohistological staining for fibrotic
nodule and type I collagen were presented in FIG. 3 and 4. As the
indicated by FIG. 3, AKF-PD treated rat has much reduced fibrotic
nodule than pyquiton and Interferon-.gamma. treated rat. Also as
shown by FIG. 4, AKF-PD treated rat has much less type I collagen
accumulation than by pyquiton and Interferon-.gamma. treated rat.
These results indicate that AKF-PD may be an effective
pharmacological agent to treat schistosoma liver fibrosis.
EXAMPLE 11
Effects of 1-(3'-Fluorophenyl)-5-Methyl-2(1H)-Pyridone (AKF-PD) on
Pulmonary Fibrosis
[0065] Bleomycin-induced rat pulmonary fibrosis is selected for
testing 5-methyl-1-(3'-fluorophenyl)-2(1H)-pyridone (AKF-PD). Male
Sprague-Dawley rats (6-8 weeks old, 180 g.about.220 g) were cared
under regular condition. The animals were randomly divided into 3
groups: sham surgical group, model disease group, and
disease/AKF-PD group. Six mg/kg/4 ml of Bleomycin was slowly
infused into the trachea of rats in the model disease and
disease/AKF-PD groups. Same amount of normal saline was infused
into the trachea of rats in the sham surgical group.
[0066] Five hundred mg/kg AKF-PD was directly administrated by
gavage to rats in the disease/AKF-PD group daily from 2 days prior
to operation through 27 days after operation. Normal saline was
used for all animals in both disease and sham surgical groups. The
animals were sacrificed at 27 days post surgery and the lung
tissues were removed for pathological sample preparation. The
HE-stained lung tissues from each rat were examined under
microscope to determine the frequency and severity of fibrotic
lesion. Evaluation of lung tissue fibrosis was carried out
according to the method of Szapiel et al., Bleomycin-induced
interstitial pulmonary disease in the nude, athymic mouse. Am. Rev.
Respir. Dis. 120:893-9(1979). As shown in Table 11, rats treated
with AKF-PD showed less severe fibrotic lesions as compared to
those in the disease group, indicating AKF-PD may be an effective
agent for treating pulmonary fibrosis disease. TABLE-US-00011 TABLE
11 Comparison of Frequency and Severity of Fibrotic Lesions
Severity of AKF-PD group Disease group Sham surgical Fibrosis Total
of 8 rats Total of 6 rats Total of 7 rats None 0 0 2 Milder 6 1 5
Moderate 1 4 0 Sever 1 1 0
EXAMPLE 12
Efficacy Comparison of 1-(3'-Fluorophenyl)-5-methyl-2(1H)-pyridone
(AKF-PD) and PIRFENIDONE In Kidney Fibrosis And Pulmonary Fibrosis
Models
Kidney Fibrosis
[0067] Anti-fibrotic effect of AKF-PD was tested on a SD rat model
for renal interstitial fibrosis induced by surgical ligation of
single side ureter. Male SD rats (180 g.about.220 g) were randomly
divided into disease model group, PFD (500 mg/kg/d) group and
AKF-PD (500 mg/kg/d) group. Under aseptic condition, all animals in
the groups of disease model, PFD and AKF-PD had a surgical
procedure for ligation of ureter. The respective drugs were
administrated by gavage to rats in the groups of PFD and AKF-PD
from one day prior to the procedure to 14 days post surgical
procedure. Normal saline was administrated in a similar fashion to
the rats in the groups of disease model. The animals were
sacrificed 14 days after surgical procedure and their kidney were
removed for pathological (HE staining) examination. Histological
scoring for interstitial compartment was done according to
Radford's method (Radford et al., Predicting renal outcome in IgA
nephropathy. J. Am. Soc. Nephrol. 8 : 199-207 (1997). As shown in
Table 12, rats treated with AKF-PD showed more reduced lesion on
interstitial tissues comparing to those in the groups of PFD
treated. Although not significantly, the data indicates that AKF-PD
may be more effective than PFD for interstitial renal fibrosis.
TABLE-US-00012 TABLE 12 Comparison of Histological Scores for
Interstitial Compartment Disease Group Model PFD AKF-PD Number of
rats 8 8 9 (n) Score 9.53 .+-. 1.75 8.06 .+-. 2.41 7.11 .+-.
1.38
Pulmonary Fibrosis
[0068] Under regular care condition, 7-10 weeks old, male,
institute of cancer research (ICR) mice, weight 28-39 g, were
randomly grouped as normal control, disease, disease/AKF-PD,
disease/PFD and disease Captopril group.
[0069] Control group was injected with normal saline once day from
day 2 to 15 days. Disease, disease/AKF-PD, disease/PFD and
disease/captopril groups were injected with bleomycin once day from
day 2 to 15. Control and disease groups were given 0.5% CMC by
gavage once a day from day 1 to day 28. 500 mg/kg of AKF-PD CMC
suspension and PD CMC suspension and 12.5 mg/kg captopril in saline
were administered to respective groups by gavage once day from day
1 to day 28. All animals were sacrificed on day 29 and their lungs
were removed for pathological (HE staining) examination.
[0070] Histological scoring for the lung tissue was done according
Szapiel's method. As shown in Table 13, both groups treated with
AKF-PD and PFD showed reduced lesion comparing to those in the
group of disease model and captopril group (a regular treatment for
fibrosis). The results indicate that AKF-PD is similar to PFD in
slowing pulmonary fibrosis. TABLE-US-00013 TABLE 13 Histological
Score for Lung Tissue Std. Group N Mean Deviation Disease 6 3.0000
.00000 Disease/Captopril 7 2.7143 0.48795 Disease/AKF-PD 11 2.0000
.77460 Disease/PFD 11 2.0909 .70065 T test disease/AKF-PD VS
disease P = 0.003 disease/AKF-PD VS disease/PDF P = 0.736
(insignificant) disease/PFD VS disease P = 0.006 Disease/Captopril
VS disease P = 0.418 (insignificant)
EXAMPLE 13
Comparison of Acute Toxicity of
1-(3'-Fluorophenyl)-5-methyl-2(1H)-pyridone (AKF-PD) and
PIRFENIDONE
[0071] Male and female Kun Ming (KM) mice weighing between 18 g-22
g were acquired from the animal facility of Hsiang-Ya Medical
College, the Central South University. Fifty Kun Ming mice were
randomly assigned into 5 groups with 5 male and 5 female mice for
each group. The animals went through fasting with a normal water
supply before starting drug treatment (either AKF-PD or
PIRFENIDONE). The drug was administrated orally by gavage. The
volume of liquid drug was 20 ml/kg body weight. The range of dosage
for AKF-PD administrated was from 1071 mg/kg to 6000 mg/kg. The
dosage difference between two adjacent doses was 1:0.65 (a dose vs
the next lower dose). All animals were maintained under a regular
condition. Acute toxic reaction and death within 14 days of post
drug treatment were recorded. Autopsy was performed on all dead
animals and visual examination was performed on all organs.
[0072] LD.sub.50 was calculated according to Bliss method. Acute
toxicity LD.sub.50 for 1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone
was 2979.89 mg/kg with a 95% confidence limit of 2402.70-3695.73
mg/kg (Table 14). LD.sub.50 for PIRFENIDONE was 955.4 mg/kg with a
95% confidence limit of 550.9-1656.7 mg/kg (Table 15). The results
of Table 15 are very close to those reported in the literature:
997.7 mg/kg (U.S. Pat. No. 5,310,562) and 1112 mg/kg(Pharmaceutical
Care and Research 5:4823(2005)). These results indicate that the
toxicity for AKF-PD is only one third of that of PIRFENIDONE (2978
vs 955). TABLE-US-00014 TABLE 14 LD.sub.50 of
1-(3'-Fluorophenyl)-5-methyl-2(1H)-pyridone Dosage in Number
Percentage Dosage log scale Number of of death LD.sub.50 and 95%
(mg/kg) (x) of Mice death (%) confidence limit 6000.0 3.7782 10 9
90 2979.89 mg/kg 3900.0 3.5911 10 7 70 (2402.70 - 3695.73) 2535.0
3.4040 10 4 40 1647.8 3.2169 10 1 10 1071.0 3.0298 10 0 0
[0073] TABLE-US-00015 TABLE 15 LD.sub.50 of PIRFENIDONE Dosage in
Number Number Percentage LD.sub.50 and 95% Dosage log scale of of
of death confidence (mg/kg) (x) Mice death (%) limit 6000 3.778 10
10 100 955.4 mg/kg 3900 3.591 10 10 100 (550.9 - 1656.7) 2535 3.030
10 8 80 1647.8 3.404 10 6 60 1071 3.217 10 5 50
EXAMPLE 14
AKF-PD: A Novel Anti-Scarring Therapy for Advanced Diabetic
Nephropathy
[0074] The study described below will determine whether the
experimental drug AKF-PD can slow kidney disease in patients with
diabetes. Diabetes can cause accumulation of proteins in the
kidneys, leading to scar formation and eventual kidney failure.
AKF-PD has been shown to reduce fibrosis in multiple experimental
models, including pulmonary fibrosis, liver sclerosis, and renal
disease. In animal models of renal diseases, AKF-PD reduces
glomerulosclerosis and interstitial fibrosis. It is anticipated
that AKF-PD may be able to slow scar formation in diabetic kidney
disease and prolong kidney function.
[0075] We will enroll 30 adult patients with type 1 or 2 diabetes
with glomerular filtration rate (GFR) between 20-75 ml/min/1.73
m.sup.2, greater than 300 mg/d of proteinuria, and blood pressure
less than or equal to 140/90 on an ACE inhibitor or an ARB.
Participants are randomly assigned to take either 1200 mg of
AKF-PD, 2400 mg of AKF-PD, or a placebo by mouth three times a day
for 1 year. They return to the clinic 2 weeks after the initial
screening visit and then every 3 months throughout the study for
fasting blood and urine tests, blood pressure measurement and
reviews of any health-related issues. Additional blood samples may
be drawn to see if AKF-PD is affecting the level of certain
proteins or other related molecules that are thought to be related
to kidney disease progression in diabetes.
[0076] Patients are asked to check their blood pressure at home at
least 3 times a week and record it in a log. A patient whose blood
pressure is greater than 130/80 must call the doctor to adjust his
or her medications. Patients may also need to monitor their blood
sugar more frequently than usual (up to 4 times a day) and possibly
give more frequent insulin injections to achieve good control of
their diabetes.
[0077] Patients are asked to collect 24-hour urine five times
during the study: at baseline, 2 weeks, 6 months, 12 months, and 54
weeks (end of study). In addition, they are seen by an eye doctor
at baseline and at the end of the study to evaluate if AKF-PD may
be beneficial for eye problems related to diabetes.
[0078] Patients will be maintained on the current standard of care
for diabetic nephropathy, including an angiotensin converting
enzyme (ACE) inhibitor and/or angiotensin receptor blocker (ARB),
antihypertensive therapy with blood pressure target of less than
130/80, and aggressive glycemic control with target hemoglobin A1C
of less than 7%.
[0079] The primary endpoint will be the change in renal function
from baseline to the end of the study period. Renal function will
be assessed by the GFR. The secondary endpoints will include the
percent change in urine albumin excretion and the levels of urine
and plasma TGF-.beta. from baseline to the end of the study
period.
[0080] Based on data from experimental animal models, it is
anticipated that AKF-PD will significantly improve renal function
and reduce TGF-.beta. levels.
* * * * *